CN110117669B - Anti-tuberculosis drug resistance detection method based on overlap extension PCR - Google Patents
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Abstract
The invention relates to an anti-tuberculosis drug resistance detection method based on overlap extension PCR, which adopts an overlap extension PCR method to connect and amplify four drug resistance related fragments of rpoB, embB, katG genes and inhA promoters into a fusion fragment with the length of about 700bp in a single tube, and only needs one sequencing reaction to obtain the drug resistance mutation information of the four fragments, thereby obtaining the drug resistance situation of a certain mycobacterium tuberculosis to three first-line anti-tuberculosis drugs. The method is popularized and applied clinically, so that the detection cost is greatly reduced, the economic burden of a patient is relieved, and good social benefits are generated. In brief, the method for detecting the drug resistance of the first-line three anti-tuberculosis drugs by using the rpoB-embB-katG-inhA fusion fragment amplified by the single-tube PCR for sequencing is simple, convenient, accurate and low in cost, and the economic burden is reduced and the detection accuracy is improved for the detection of clinical drug-resistant tuberculosis.
Description
Technical Field
The invention belongs to the technical field of genes, and relates to a method for detecting drug resistance of three anti-tuberculosis drugs at one time based on overlap extension PCR.
Background
Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis (Mtb), and is a major public health problem seriously harming human health. In recent years, the drug resistance of mycobacterium tuberculosis gradually worsens, and the situation of drug-resistant tuberculosis is very severe, so the importance of drug-resistant tuberculosis diagnosis is increasingly prominent. While culture-based methods remain the gold standard for diagnosing drug-resistant tuberculosis, traditional drug sensitivity tests for detecting Mtb drug resistance can take up to several weeks, delay patient treatment and can result in new drug resistance, be labor intensive, and can pose serious biohazards to laboratory workers. Rapid diagnosis of drug resistance in tuberculosis patients is therefore increasingly gaining importance.
The rapid genotype drug resistance detection method can reduce to several manual steps, is more suitable for instant detection, and can remarkably expand the application of the rapid genotype drug resistance detection method in the population with insufficient medical services. At present, the detection methods of genotype drug resistance which are applied more at home and abroad mainly comprise a real-time fluorescence PCR method, a linear probe hybridization method and a gene chip method. The principle of the genotype drug resistance detection methods is based on the research on the correlation of Mtb gene sequence mutation and drug resistance of the Mtb gene sequence mutation to various drugs. Multiple studies have shown that in most cases resistance of Mtb to the major anti-TB drugs is mainly associated with about 25 mutations in the six genes and promoter regions. About 90% of isoniazid resistance cases can detect katG gene codon 315 and inhA promoter nucleotide-8 to-15 mutation. Approximately 90% of fluoroquinolone resistance cases can detect the occurrence of mutations at codons 88 to 94 of the gyrA gene, with mutations at codons 500 and 538 to 540 of the gyrB gene accounting for a small percentage of cases. Mutations at the sites 1401 and 1402 of the rrs gene identified about 80% of amikacin resistance cases, with the same rrs mutation plus a mutation at nucleotides-8 to 37 of the eis promoter-associated kanamycin resistance of about 80%. 47% -89% of ethambutol-resistant Mtb has a 306-bit codon mutation of an arabinose transferase coding gene embB gene.
Among the methods based on real-time fluorescence PCR, geneXpert MTB/RIF and melting curve analysis are the main ones. However, the resistance portion of the GeneXpert MTB/RIF assay only provided information about rifampicin resistance, since it was difficult to detect all clinically drug-resistance-associated mutations in 6 independent genes or promoter regions in a single reaction tube. The main disadvantage of the melting curve analysis method is that melting curve peaks of a plurality of reaction probes are overlapped, which increases the complexity of the melting curve analysis; in addition the signal sensitivity is relatively low. The linear probe and the gene chip technology are relatively complicated to operate for detecting the drug resistance of Mtb. These methods are similar to the GeneXpert MTB/RIF assay, and one kit can only detect drug resistance of one drug.
In conclusion, the genotype drug resistance detection methods have the advantages of high efficiency, high speed and the like, can quickly diagnose tuberculosis and partial drug resistance conditions thereof, but have relatively high detection cost. The expensive price makes the economic burden on patients aggravate in poor regions. And in the case that a small part of detection results do not accord with sequencing results, false drug resistance results are wrong when synonymous mutation exists in a probe binding site in a coding region of a drug resistance related gene.
Disclosure of Invention
In view of the above, the present invention provides a method for detecting three anti-tuberculosis drug resistance at a time based on overlap extension PCR.
In order to achieve the purpose, the invention provides the following technical scheme:
1. the detection primers comprise four fragment PCR primers which are designed by taking a DNA sequence of a mycobacterium tuberculosis standard strain H37Rv as a template and comprise common drug resistant mutation sites of rpoB, embB, katG genes and inhA promoters, and are specifically as follows:
rpobF:5'-GTACGGTCGGCGAGCTGATCCA-3' as shown in SEQ ID No. 1;
rpobR：
5'-CACCGGGTGCACGTCGCGGACCTCCAGCCCGGCACGCTCACGTGACAGAC-3' as shown in SEQ ID No. 2;
embF:5'-GGAGGTCCGCGACGTGCACCCGGTGATATTCGGCTTCCTGCTC-3' as shown in SEQ ID No. 3;
embR:5'-ACGGAAGGGATCCTCCGGGCTGCCGAACCAGCGGAAATAGTTGGA-3' as shown in SEQ ID No. 4;
katF:5'-CGGCAGCCCGGAGGATCCCTTCCGTATGGCACCGGAACCGGTAA-3' as shown in SEQ ID No. 5;
katR：
5'-ACGCAAGCGCCAGCAGGGCTCTTCGTCAGCTCCCACTCGTAGCCGTACA-3' as shown in SEQ ID No. 6;
inhF：
5'-GACGAAGAGCCCTGCTGGCGCTTGCGTAACCCCAGTGCGAAAGTTCCCG-3' as shown in SEQ ID No. 7;
inhR:5'-GGACTGAACGGGATACGAATGG-3' as shown in SEQ ID No. 8.
As one of the preferable technical proposal, the design method of three pairs of overlapping extension primers comprises the following steps: the 5' ends between rpobR and embF, between embR and katF and between katR and inhF respectively contain a section of overlapping complementary sequence of 25bp to 27bp, and the three sections of overlapping complementary sequences are different from each other.
2. The kit comprises the detection primers, wherein the concentration ratio of the primers is rpobF: rpobR: embF: embR: katF: katR: inhF: inhR =100:5:1:5:5:1:5:100 to 60:2:2:1:1:2:2:60, selecting the optimal proportion of 30:1:1:1:1:1:1:30, of a nitrogen-containing gas; the final concentrations of rpobF and inhR are 0.5-1.0. Mu.M.
3. The detection primer or the kit is applied to the detection of the drug resistance of the antituberculosis drug.
4. The method for detecting the drug resistance of the antituberculous drug based on the overlap extension PCR, which is realized by utilizing the detection primer or the kit, comprises the following specific steps:
(1) DNA extraction: extracting DNA of mycobacterium tuberculosis as a template;
(2) Constructing a PCR reaction system:
10×BUFFER 5.0μl，
dNTPs 1.0μl(2.5mM each)，
hot start Taq enzyme 0.2. Mu.l (5U/. Mu.l),
mu.l of primer mixture (rpobF and inhR concentration 10. Mu.M, primers mixed in the above ratio),
mu.l of the template DNA was prepared,
water was added to make 50. Mu.l in total.
(3) Carrying out PCR reaction;
(4) Sequencing analysis: and sequencing the rpoB-embB-katG-inhA fusion fragment, and comparing and analyzing the sequence with the DNA sequence of a standard strain H37Rv to obtain the amino acid sequence and related drug-resistant mutation information.
As one of the preferable technical scheme, in the step (1), the specific method for extracting the DNA comprises the following steps: extracting DNA of a sample by using a DNA extraction kit for mycobacterium tuberculosis according to the instruction operation or a boiling method to be used as a template.
As one of the preferable technical schemes, in the step (3), an ABI7500 real-time fluorescent quantitative PCR instrument (a common instrument capable of setting touchdown annealing temperature) is used for carrying out PCR reaction, and the specific process is as follows: the first step is as follows: 5 minutes at 95 ℃; the second step is that: 10 seconds at 95 ℃; the third step: 68 ℃ per cycle, 0.5 ℃ for 10 seconds, fourth step: 30 seconds at 72 ℃, and circulating for 30 times from the second step to the fourth step; the fifth step: 95 ℃ for 10 seconds, sixth step: 59 ℃ for 10 seconds, seventh step: 30 seconds at 72 ℃, and 40 times of circulation from the fifth step to the seventh step.
As one of the preferred embodiments, in the step (4), the rpoB-embB-katG-inhA fusion fragment is sequenced using a nucleotide sequence such as TBseq shown in the sequencing primer SEQ ID NO. 9;
TBseq:5'-ACCAGATCCGGGTCGGCATG-3' as shown in SEQ ID No. 9.
The invention has the beneficial effects that:
the invention adopts an overlap extension PCR method, connects and amplifies four drug-resistant related fragments of rpoB, embB, katG genes and inhA promoters into a fusion fragment with the length of about 700bp in a single tube, and obtains the drug-resistant mutation information of the four fragments only by one sequencing reaction, thereby obtaining the drug-resistant conditions of a certain mycobacterium tuberculosis to three first-line anti-tuberculosis drugs, namely rifampicin, isoniazid and ethambutol. The method is used for detecting more than 100 clinical specimens, and the reliability of the result is verified by comparing with a culture method drug sensitivity test and GeneXpert. The method is clinically popularized and applied, so that the detection cost is greatly reduced, the economic burden of a patient is relieved, and good social benefits are generated. In brief, the method for detecting the drug resistance of the first-line three anti-tuberculosis drugs (rifampicin, isoniazid and ethambutol) by using the rpoB-embB-katG-inhA fusion fragment amplified by the single-tube PCR for sequencing is simple, convenient, accurate and low in cost, and the economic burden is reduced and the detection accuracy is improved for the detection of clinical drug-resistant tuberculosis.
The invention connects and amplifies four drug-resistant related fragments of rpoB, embB, katG gene and inhA promoter into a fusion fragment with the length of about 700bp in a single tube. The fusion fragment can be sent to a sequencing company for sequencing, and the sequencing result can be obtained in one day generally. And the fragment with the length of about 700bp only needs one sequencing reaction to obtain the drug resistance mutation information of the four fragments, so that the drug resistance conditions of the specimen Mtb to three first-line antitubercular drugs of rifampicin, isoniazid and ethambutol are obtained. The detection accuracy of the method is higher than that of the GeneXpert and the melting curve method, and the detection cost is greatly reduced. Moreover, the PCR fragment has no biological hazard of Mtb bacteria, and the potential biological safety hazard can be avoided when the PCR fragment is delivered to a sequencing company than when a bacteria-containing sample or the Mtb strain is delivered to a laboratory with drug resistance detection capability. The successful establishment and the clinical popularization and application of the method can greatly reduce the economic burden of patients, improve the biological safety of detection and generate good social benefits.
The main advantages of the present invention can be summarized as the following two points:
1) Simplified PCR and sequencing steps: amplifying rpoB, embB, katG and inhA four fragments from four tubes respectively, sequencing for four reactions respectively, and simplifying to a single tube only for amplifying one fusion fragment and sequencing for one reaction; the four fragments to be amplified are connected into one fragment by using overlap extension PCR, so that the troublesome operation of respectively carrying out the four PCRs is reduced, and simultaneously, the four sequencing reactions of the four fragments are reduced to one sequencing reaction, so that the detection labor intensity is reduced, and the detection cost is greatly reduced.
2) The accuracy of drug resistance detection is improved: both the probe method and the melting curve method are methods for detecting whether or not a sequence has a mutation, but cannot detect what mutation is. The invention can directly detect a specific mutation mode and avoid the phenomenon that the probe method and the melting curve method falsely report the synonymous mutation of rpoB and embB genes as drug-resistant mutation. The sequencing method is a gold standard compared with other methods such as a probe method, a melting curve method, a chip method and the like required by WHO, and the detection accuracy is improved by detecting the drug-resistant mutation by using the sequencing fusion fragment.
Drawings
In order to make the object, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings for explanation:
FIG. 1 is a schematic diagram of the detection process of the present invention;
FIG. 2 is a gel electrophoresis diagram of a PCR product of a part of the specimen, wherein M is DNA Marker DL2000plus,1-20 are amplification products of the specimen, the size is close to 750bp, and N is a negative control.
Detailed Description
The preferred embodiments of the present invention will be described in detail below.
Example (b):
the method for detecting the drug resistance of the antituberculous drug based on the overlap extension PCR comprises the following specific steps:
1. overlap extension PCR amplification of fusion fragment (see FIG. 1 for principle)
(1) DNA extraction:
extracting DNA of a sample by using a DNA extraction kit for mycobacterium tuberculosis according to the instruction operation or a boiling method to be used as a template; (physiological saline as a negative control, since there is no natural rpoB-embB-katG-inhA fusion fragment, no positive control)
(2) Constructing a PCR reaction system:
10×BUFFER 5.0μl，
dNTPs 1.0μl(2.5mM each)，
hot start Taq enzyme 0.2. Mu.l (5U/. Mu.l),
mu.l of primer mixture (rpobF and inhR concentration 10. Mu.M, primers mixed in the above ratio),
mu.l of the template DNA was added,
water was added to make 50. Mu.l in total.
3) PCR reaction procedure:
taking ABI7500 as an example, the first step: 5 minutes at 95 ℃; the second step is that: 10 seconds at 95 ℃; the third step: 68 ℃ per cycle, 0.5 ℃ for 10 seconds, and a fourth step: 30 seconds at 72 ℃, and 30 times of circulation from the second step to the fourth step; the fifth step: 95 ℃ for 10 seconds, sixth step: 59 ℃ for 10 seconds, seventh step: 30 seconds at 72 ℃, and circulating for 40 times from the fifth step to the seventh step;
2. sequencing and mutation analysis
The sequencing primer is TBseq:5'-ACCAGATCCGGGTCGGCATG-3'; as shown in SEQ ID NO. 1;
the rpoB-embB-katG-inhA fusion fragment was sequenced using primer TBseq (Chongqing division, biotech, inc., of Beijing Ongskaceae). And comparing and analyzing the detected sequence with the DNA sequence of the standard strain H37Rv to obtain the amino acid sequence and related drug-resistant mutation information. The rpoB-embB-katG-inhA fusion DNA sequence with the standard strain H37Rv as a template is as follows:
CAAAACCAGATCCGGGTCGGCATGTCGCGGATGGAGCGGGTGGTCCGGGAGCGGATGACCACCCAGGACGTGGAGGCGATCACACCGCAGACGTTGATCAACATCCGGCCGGTGGTCGCCGCGATCAAGGAGTTCTTCGGCACCAGCCAGCTGAGCCAATTCATGGACCAGAACAACCCGCTGTCGGGGTTGACCCACAAGCGCCGACTGTCGGCGCTGGGGCCCGGCGGTCTGTCACGTGAGCGTGCCGGGCTGGAGGTCCGCGACGTGCACGCGGTGATATTCGGCTTCCTGCTCTGGCATGTCATCGGCGCGAATTCGTCGGACGACGGCTACATCCTGGGCATGGCCCGAGTCGCCGACCACGCCGGCTACATGTCCAACTATTTCCGCTGGTTCGGCAGCCCGGAGGATCCCTTCGCGTATGGCACCGGAACCGGTAAGGACGCGATCACCAGCGGCATCGAGGTCGTATGGACGAACACCCCGACGAAATGGGACAACAGTTTCCTCGAGATCCTGTACGGCTACGAGTGGGAGCTGACGAAGAGCCCTGCTGGCGCTAGCGTAACCCCAGTGCGAAAGTTCCCGCCGGAAATCGCAGCCACGTTACGCTCGTGGACATACCGATTTCGGCCCGGCCGCGGCGAGAYGATAGGTTGTCGGGGTGACTGCCACAGCCACTGAAGGGGCCAAACCCCCATTCGTATCCCGTTCAGTCC
the sequence of rpoB-embB-katG-inhA fusion DNA fragment with the standard strain H37Rv as a template was converted into the following amino acid sequence (no amino acid sequence of inhA promoter):
QNQIRVGMSRMERVVRERMTTQDVEAITPQTLINIRPVVAAIKEFFGTSQLSQFMDQNNPLSGLTHKRRLSALGPGGLSRERAGLEVRDVHAVIFGFLLWHVIGANSSDDGYILGMARVADHAGYMSNYFRWFGSPEDPFAYGTGTGKDAITSGIEVVWTNTPTKWDNSFLEILYGYEWELTKSPAGA
sequence annotation:
rpoB(1-273bp)-embB(274-420bp)-katG(421-564bp)-inhA(565-722bp)
embB 306 site: 346-348bp,116 amino acid residue
katG 315 site: 457-459bp,153 amino acid residue
inhA promoter (-15) site: 653bp, (-8) site: 660bp of
The correspondence between the mutation sites of the common rpoB amino acid residues and the alignment of the standard strain H37Rv is shown in Table 1.
TABLE 1 alignment sequence mapping
| Position of rpoB amino acid residue | Amino acid residue positions of aligned sequences | Alignment of sequence DNA sites |
| 511 | 51 | 151-153 |
| 513 | 53 | 157-159 |
| 515 | 55 | 163-165 |
| 516 | 56 | 166-168 |
| 518 | 58 | 172-174 |
| 519 | 59 | 175-177 |
| 526 | 66 | 196-198 |
| 531 | 71 | 211-213 |
| 533 | 73 | 217-219 |
3. Study of clinical applications
108 clinical mycobacterium tuberculosis specimens are collected, DNA is respectively extracted as a template, and the fusion fragments of the specimens are amplified by the overlap extension PCR method established by the invention (figure 2). And (3) sequencing the fused fragments obtained by the amplification of each specimen, and analyzing the drug resistance mutation of the fused fragments through a sequence to obtain the results of the three first-line antituberculosis drugs. Then compared with the results of the drug sensitivity test by the culture method, the results are shown in tables 2 to 4.
TABLE 2 detection of rifampicin resistance by the method and culture method of the present invention
TABLE 3 detection of isoniazid drug resistance by the method and culture method of the present invention
TABLE 4 detection of ethambutol resistance by the methods and culture methods of the present invention
Note: s represents sensitivity; r represents drug resistance
From tables 2-4, the consistency of rifampicin, isoniazid and ethambutol resistance detected by the method and the culture method is respectively 94.4%, 94.4% and 76.9%, and the coincidence rate of the genotype resistance detection method and the phenotype is achieved.
The results of 56 specimens respectively detected by the method of the invention and the GeneXpert method are shown in Table 5, and the consistency of rifampicin drug resistance detected by the two methods reaches 100%.
TABLE 5 detection of rifampicin resistance by the method of the invention and GeneXpert
Finally, it is noted that the above-mentioned preferred embodiments illustrate rather than limit the invention, and that, although the invention has been described in detail with reference to the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as defined by the appended claims.
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Claims (3)
1. The detection primer is characterized by comprising four fragment PCR primers which are designed by taking a DNA sequence of a mycobacterium tuberculosis standard strain H37Rv as a template and contain common drug-resistant mutation sites of rpoB, embB, katG genes and inhA promoters, and specifically comprises the following steps:
rpobF:5'-GTACGGTCGGCGAGCTGATCCA-3' as shown in SEQ ID No. 1;
rpobR：
5'-CACCGGGTGCACGTCGCGGACCTCCAGCCCGGCACGCTCACGTGACAGAC-3' as shown in SEQ ID No. 2;
embF：
5'-GGAGGTCCGCGACGTGCACCCGGTGATATTCGGCTTCCTGCTC-3' as shown in SEQ ID No. 3;
embR：
5'-ACGGAAGGGATCCTCCGGGCTGCCGAACCAGCGGAAATAGTTGGA-3' as shown in SEQ ID No. 4;
katF：
5'-CGGCAGCCCGGAGGATCCCTTCCGTATGGCACCGGAACCGGTAA-3' as shown in SEQ ID No. 5;
katR：
5'-ACGCAAGCGCCAGCAGGGCTCTTCGTCAGCTCCCACTCGTAGCCGTACA-3' as shown in SEQ ID No. 6;
inhF：
5'-GACGAAGAGCCCTGCTGGCGCTTGCGTAACCCCAGTGCGAAAGTTCCCG-3' as shown in SEQ ID No. 7;
inhR:5'-GGACTGAACGGGATACGAATGG-3' as shown in SEQ ID No. 8.
2. A kit comprising the detection primers of claim 1, wherein the concentration ratio of each primer is rpobF: rpobR: embF: embR: katF: katR: inhF: inhR =30:1:1:1:1:1:1:30, of a nitrogen-containing gas; the final concentrations of rpobF and inhR are 0.5-1.0 mu M.
3. The use of the detection primers as defined in claim 1 or the kit as defined in claim 2 for the detection of resistance of mycobacterium tuberculosis to rifampicin, isoniazid, ethambutol, three antitubercular drugs, said use being for non-diagnostic purposes.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2009268360A (en) * | 2008-04-30 | 2009-11-19 | Yamaguchi Univ | Primer for producing fused dna fragment and method for producing fused dna fragment using the same |
| CN106755418A (en) * | 2016-12-24 | 2017-05-31 | 广州艾基生物技术有限公司 | A kind of extron assembles sequence measurement |
| CN109777867A (en) * | 2018-12-29 | 2019-05-21 | 广州凯普医药科技有限公司 | A kind of method that Overlap extension PCR combination Sanger sequencing detects deaf susceptibility gene mutation |
-
2019
- 2019-06-04 CN CN201910481695.2A patent/CN110117669B/en active Active
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|---|---|---|---|---|
| JP2009268360A (en) * | 2008-04-30 | 2009-11-19 | Yamaguchi Univ | Primer for producing fused dna fragment and method for producing fused dna fragment using the same |
| CN106755418A (en) * | 2016-12-24 | 2017-05-31 | 广州艾基生物技术有限公司 | A kind of extron assembles sequence measurement |
| CN109777867A (en) * | 2018-12-29 | 2019-05-21 | 广州凯普医药科技有限公司 | A kind of method that Overlap extension PCR combination Sanger sequencing detects deaf susceptibility gene mutation |
Non-Patent Citations (2)
| Title |
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| An application of competitive reporter monitored amplification (CMA) for rapid detection of single nucleotide polymorphisms (SNPs);Juliane Havlicek等;《PLoS One》;20170829;第12卷(第8期);第2页第3段、第4页第5段至第5页第1段、附加表1 * |
| Juliane Havlicek等.An application of competitive reporter monitored amplification (CMA) for rapid detection of single nucleotide polymorphisms (SNPs).《PLoS One》.2017,第12卷(第8期),第1-13页. * |
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